Results: The Heterozygous alleles G/A in factor V gene was 8.0% in all cases ... Key Words: Factor V Leiden G1691A, Factor II G20210A, RSA, Sudanese ...
Point Mutation in Factor V Leiden G1691A and Factor II G20210A
JIIMC 2016 Vol. 11, No.3
ORIGINAL ARTICLE
Point Mutation in Factor V Leiden G1691A and Factor II G20210A and Effect on Coagulation Profile and Frequency of Recurrent Spontaneous Abortions among Sudanese Women Asaad Mohammed Ahmed Abd Allah Babker1, Fath Elrahman Mahdi Hassan Gameel2, Salaheldein Gumaa Elzaki3, Amanda G Elgoraish4, Lienda Bashier Eltayeb5, Hisham Ali Waggiallah6
ABSTRACT Objective: To investigate the effect of point mutation in FV Leiden G1691A and FII G20210A gene on coagulation and recurrent spontaneous abortion (RSA) among Sudanese women. Study Design: This was retrospective case control study. Place and Duration of Study: The study was carried out from Aug 2012 to Dec 2014 at Omdurman Maternal Hospital, Sudan. Materials and Methods: The study included hundred pregnant females with a history of recurrent spontaneous abortion as (case group) and ninety five healthy reproductive Sudanese women as (control group). The data was collected with the help of structured questionnaire and direct interview to collect information. Identification of point mutation in factor V Leiden G1691A and factor II G20210A gene by polymerase chain reaction was performed; Coagulometer was used for the measurement of PT and APTT. Odds Ratio and the 95% confidence interval (95%CI) were calculated for the presence of mutation between cases and controls and analyzed by SPSS program, version 17.0. Results: The Heterozygous alleles G/A in factor V gene was 8.0% in all cases related with three, four and five times of recurrent abortion and 6% was found in control group. Heterozygous alleles of factor II G/A Prothrombin time (PT) and partial thromboplastin time (PTT) in women with Recusant Spontaneous Abortion (RSA) were not affected significantly (P=0. 93 and P=0.69). Conclusion: Based upon the results it is concluded that the point mutation in factor V Leiden G1691A and factor II G20210A might play a role in recurrent spontaneous abortion loss among Sudanese women. However these point mutations do not affect the coagulation profile. Key Words: Factor V Leiden G1691A, Factor II G20210A, RSA, Sudanese Pregnant Women.
Introduction Recurrent pregnancy loss (RPL) is defined as three or th more consecutive pregnancy losses before the 24 1 Department of Clinical Laboratory Al-Ghad International Colleges for Health Sciences Al-Madinah Al-Munawarah, Saudia Arabia 2 Department of Hematology and Immunohaematology College of Medical Laboratory Science Sudan University of Science and Technology Khartoum, Sudan 3,4 Department of Epidemiology Tropical Medicine Research Institute National Centre for Research, Khartoum, Sudan 5 Department of Medical Parasitology Faculty of Medical Laboratory Sciences Omdurman Islamic University, Khartoum, Sudan 6 Department of Clinical Laboratory Al-Ghad International Colleges for Health Sciences Al Riyadh, Saudia Arabia
Correspondence: Dr. Hisham Ali Waggiallah Department of Clinical Laboratory Al-Ghad International Colleges for Health Sciences Al Riyadh, Saudia Arabia Funding Source: NIL ; Conflict of Interest: NIL Received: Mar 17, 2016; Revised: Apr 21, 2016 Accepted: Aug 29, 2016
week of gestation .The modern definition, however, is the spontaneous loss of 2 or more consecutive 1 pregnancies before 20 weeks of gestation. Recurrent pregnancy loss is an experience which can be very painful for the couple. Most of the miscarriages occur in the first trimester and affect about 15% of all recognized pregnancies.2 RPL has many possible causes that can be categorized as genetic abnormalities, hormonal and metabolic disorders, uterine anatomic abnormalities, i nfe c t i o u s ca u s e s , i m m u n e d i s o rd e rs a n d 3 thrombophilic disorders. Hereditary thrombophilias are a group of genetic disorders of blood coagulation resulting in a hypercoagulable state, which in turn can result in abnormal placentation. Early in pregnancy this may manifest as spontaneous loss.4 Factor V Leiden (FVL) and prothrombin gene (G20210A) mutations are the most common types of hereditary thrombophilias. Most carriers of this mutation do not develop any clinical signs and remain undiagnosed because these conditions result 108
JIIMC 2016 Vol. 11, No.3
in a small absolute risk of clinically significant thrombosis. 5 Factor V Leiden is a single point mutation involving a guanine to adenine transition at position 1691 in exon 10 of the factor V gene, which leads to the synthesis of a variant factor V molecule.6 The prothrombin G20210A mutation involves guanine to adenine substitution at nucleotide 20210 of the prothrombin gene.7 FV Leiden and factor II G20210A mutations are associated with increased production of thrombin and risk of venous 8 thrombosis. Also Factor V Leiden mutation is found to be the most common inherited thrombotic risk factor associated with RPL its frequency in whites varies from 3% to 8% and 1 in 1000 are homozygous. It is rare in African Americans, Asians and Native 9 Americans. The incidence of genetic prothrombotic mutations in women with unexplained pregnancy loss was examined in various studies: some of these 10,11 studies supported the association. While others 12,13 re p o r te d n o a s s o c i at i o n . T h e p re s e nt retrospective case control study was conducted to evaluate the FV Leiden G1691A and FII G20210A mutations and their affect on some coagulation profiles (PT and PTT) among women with a history of three or more consecutive pregnancy losses and healthy controls. This is the first study that investigated FV Leiden G1691A and FII G20210A alleles and genotype distributions in the Sudanese females with habitual RPL.
Materials and Methods This was retrospective case control study. The genomic DNA samples of one hundred and ninety five Sudanese women who recruited and followed at Omdurman Maternal Hospital were screened from Aug 2012 to Dec 2014. One hundred cases having a history of RPL were compared with ninety five healthy reproductive Sudanese women as control group with a history of two or more successful live birth. Cases and controls were tested for the FV Leiden G1691A and FII G20210A. Genomic DNA was extracted from 3–5 ml of EDTA anti-coagulated blood by salting.14 DNA was extracted from the blood samples using Master pure DNA purification kit for blood GF-1 Blood DNA Extraction Kit, 50 PREPS (cat. No. GF-BD-050, Vivantis Technologies Sdn. Bhd., Malaysia). FV Leiden G1691A and FII. a 345-bp genomic DNA fragment encompassing a part of the prothrombin gene that contains the mutation was 109
Point Mutation in Factor V Leiden G1691A and Factor II G20210A
amplified by PCR using specific primers Forward (5'TCT AGA AAC AGT TGC CTG GC-3') and Reverse primer (5'ATA GCA CTG GGA GCA TTG AAG C-3). And 267-basepair (bp) segment of the factor V gene was amplified used specific forward primer (5'TCA GGC AGG AAC AAC ACC AT-3') and reverse primer 5'GGT TAC TTC AAG GAC AAA ATA CCT GTA AAG C T3. The reaction program was as follows: Denaturation at 94°C for 30 seconds, annealing at 51°C for 30 seconds, extension at 72°C for 30 seconds 15 for 35 cycles and 72 °C for 5 minute. A master mix was prepared by adding Nuclease free water,10x b u f fe r, d N T P,t o w p r i m e rs , M g c l 2 ,Ta q D N A polymerase and DNA, the mixture was loaded into thermocycler according to the specific Temperature profile. The working solution of 1X TBE is prepared from the stock solution (1 L) which contains the following: 89 mM Tris base (108 gm), 89 mM boric acid (55 gm) 40 ml of 0.5M EDTA, adjust pH to 8.0.1.5% agarose was prepared from 1x TBE, and 5µl PCR products was loaded by mixing PCR products with 1µl loading dye, run on the gel for 30 mins and visualized on UV transllimantor. Factor V Digested with 10 µl of DNA restriction enzyme MnI1 at 37°Cfor 18 h, subjected to 2% low melting point agarose and Prothrombin product (10 μL) was digested with 20 U of Hind III, at 37°C for 16 h, and loaded into 2% low melting point agarose gel, eletropherosed at 90 volts for 60 mins . Data were statistically described in terms of mean ± standard deviation (± SD), median and range, or frequencies (number of cases) and percentages where appropriate. Odds Ratio (OR) and the 95% confidence interval (95%CI) were calculated for the presence of mutation between cases and controls and analyzed by SPSS prograrmme (version: 17.0). Data were analyzed using the Chi-square test to compareson the prevalence of MTHFR mutation between patients and controls (The test considered significant when P.value
